Note: Descriptions are shown in the official language in which they were submitted.
INJECTABLE COMPOSITION COMPRISING CYTOLYTIC
COMPOUND IN GEL, GEL-FORMING SOLUTION OR
GEL-FORMING SUSPENSION FOR REDUCTION OF FAT
Background of the Invention
Technical Field
10011 The present invention relates to preparation of injectable compositions.
More
particularly, the present invention relates to an injectable composition
comprising cytolytic
compound in gel, gel-forming solution or gel-forming suspension for reduction
of fat; use or
method for the reduction or removal of localized fat by administering the
injectable composition
of the invention. In particular, the injectable composition of the invention
may be in the form of
gel during or after injection.
Description of Related Art
10021 Submental fat or double chin is usually resistant to diet or exercise,
therefore the
non-surgical fat removal injection with the active ingredient deoxycholic acid
has become a
novel treatment to reduce submental fat.
10031 Deoxycholic acid (DCA) is a secondary bile acid which can emulsify and
solubilize fat
for digestion and absorption in the intestine. Its salt, sodium deoxycholate
(DCA-Na), is an
anionic detergent commonly used to lyse cells. DCA is a TGR5 agonist (Takeda
G-protein-coupled receptor 5, GPBAR1) and the activation of TGR5 was found to
reduce
obesity in high-fat-diet fed animals. DCA is predicted to lyse adipocytes and
resulting in fat
reduction. However, cytolysis will attract inflammatory cells such as
macrophages and
monocytes to remove destroyed fat cells. Patients who received deoxycholic
acid treatment will
commonly experience swelling, pain, numbness, redness, and areas of hardness
in the treatment
due to the inflammation, and thus the interval between each treatment is long
(around a month)
as the histological evidence showed that posttreatment inflammation was
largely resolved by this
time. DCA-Na can form hydrogels under low pH, mixing with
tris(hydroxymethyDaminomethane (TRIS) buffer or mixing with polymers and an
amino acid,
L-aspartic acid. The release of additional solutes on the DCA-Na/TRIS
hydrogels was found to
be sustained, thus should be a suitable drug deliver and release platform.
Although a study
showed that adding with amino acids L-lysine and L-arginine, but not glycine
and L-a-alanine,
weakened their hydrogel formation, we successfully constructed a DCA-Na gel
system by
mixing with basic amino acids, such as L-lysine, L-arginine and L-histidine,
and/or organic acid,
such as acetic acid.
CA 03220724 2023- 11- 28
[004] Studies have shown that, after injection of deoxycholic acid solution,
deoxycholic acid
permeates into fat tissue more than 1 centimeter. A fat tissue ball with
diameter more than
2-centimeter goes into inflammation reaction. When deoxycholate gel solution
is injected into fat
tissue, only fat cells surrounding deoxycholate gel are destroyed gradually
during 7-days
slow-releasing of deoxycholate. Inflammation reaction is limited to this less
than 2 millimeters
thin layer of fat cells surrounding deoxycholate gel. Total volume of
inflammatory fat tissue is
less than 10% of traditional cytolytic injection. Finally, a cavity with
volume proportional to
injected dose of deoxycholate appears in fat tissue, and disappears within 2-3
weeks.
[005] Thus, a slow-releasing deoxycholic acid, or its salt sodium deoxycholate
(DCA-Na) gel
at the injected sites is expected, which is constructed by mixing with basic
amino acids, such as
L-lysine, L-arginine and L-histidine, and/or organic acid, such as acetic
acid, so that the cytolytic
reaction could be limited to deoxycholate-immersed fat cells surround gel
surface.
Anti-inflammatory drug or local anesthetic could also be added to the
injections during the
treatment to reduce inflammation and pain. Moreover, we also aimed to increase
the
concentration of DCA-Na so that cytolysis could be more effective, thus
patients can complete
their treatment within fewer treatment sessions. Taken together, the mixture
of DCA-Na, basic
amino acid and/or organic acid, and anti-inflammatory drug and/or local
anesthetic should
reduce or remove fat, and effectively reduce the adverse effects, and reduce
the interval between
each treatment and the whole treatment process. The compositions of DCA-Na
injections will
preferably form a gel-like appearance later than 5 minutes and before 120
minutes after mixing.
Summary of the Invention
[006] The present invention provides an injectable composition of cytolytic
compound,
preferably deoxycholic acid or a salt thereof, more preferably DCA-Na, in the
form of gel,
gel-forming solution or gel-forming suspension. The injectable composition may
be used for
reducing or removing localized fat, and have less adverse effects and
relatively short treatment
process.
[007] In one aspect, the invention provides an injectable composition
comprising cytolytic
compound in gel, gel-forming solution or gel-forming suspension for reduction
of fat,
comprising:
a cytolytic compound as a first component; and
a pharmaceutically acceptable excipient.
[008] Preferably, the cytolytic compound is deoxycholic acid or a salt
thereof.
CA 03220724 2023- 11- 28
[009] More preferably, the cytolytic compound is DCA-Na, and the injectable
composition
further comprises a second component selected from one or more of a basic
amino acid or an
organic acid.
[010] In some embodiments, the concentration of DCA-Na is 7-51 mg/mL.
[011] In some embodiments, the basic amino acid is L-lysine.
[012] In one embodiment, the concentration of L-lysine is 11-145 mg/mL.
[013] In another embodiment, the pH of L-lysine before mixing is <8.0, and the
pH of the
injectable composition is 6.45-7.75.
[014] In another embodiment, the injectable composition further comprises an
anti-inflammatory drug as a third component.
[015] Preferably, the anti-inflammatory drug is aspirin.
[016] More preferably, the concentration of aspirin is 14-100 mg/mL.
[017] Preferably, the injectable composition further comprises a local
anesthetic as a fourth
component.
[018] More preferably, the local anesthetic is Lidocaine.
[019] More preferably, the concentration of Lidocaine is 2.5-6.5 mg/mL.
[020] Preferably, the anti-inflammatory drug is Dexamethasone Sodium Phosphate
(DSP).
[021] More preferably, the pH of the injectable composition is 6.45-7.40.
[022] More preferably, the concentration of DSP is not more than 1 mg/mL.
[023] In some embodiments, the basic amino acid is L-histidine.
[024] Preferably, the concentration of L-histidine is 1.4-11.5 mg/mL.
[025] In some embodiments, the basic amino acid is L-arginine.
[026] Preferably, the concentration of L-arginine is 115-143 mg/mL.
[027] In some embodiments, the organic acid is acetic acid.
[028] Preferably, the concentration of acetic acid is 46-143 x i0 %.
[029] In other embodiments, the injectable composition further comprises
saline.
[030] In other embodiments, the injectable composition is in the form of a
gel, preferably
during and after injection.
[031] In another aspect, the invention provides use of the injectable
composition described
above, for the reduction or removal of localized fat in a subject in need
thereof, wherein the
injectable composition is subcutaneously injected into a subcutaneous
injection site of the
subject.
[032] In another embodiment, the subcutaneous injection site is the localized
fat within face,
chin, arm, waist, abdomen or thigh of the subject.
CA 03220724 2023- 11- 28
[033] In another aspect, the invention provides use of the injectable
composition described
above, for production of a medicine for the reduction or removal of localized
fat.
[034] In another aspect, the invention provides a method for reducing or
removing localized
fat in a subject in need thereof, comprising administering, preferably
subcutaneously injecting to
the subject, an effective amount of the injectable composition described
above.
[035] In another embodiment, the subject is human.
[036] In another embodiment, the injectable composition is administered,
preferably
subcutaneously injecting to the localized fat within face, chin, arm, waist,
abdomen or thigh of
the subject.
[037] The injectable composition of the invention may also comprise saline,
and may be in the
form of gel during or after injection.
BRIEF DESCRIPTION OF THE FIGURES
10381 FIG. 1 Appearances of the mixture of DCA-Na solutions and (a) 100 mg/mL,
(b) 200
mg/mL, (c) 300 mg/mL, (d) 400 mg/mL or (e) 500 mg/mL L-lysine solutions.
10391 FIG. 2 Appearances of the mixture of DCA-Na solutions and (a) 200 mg/mL
or (b) 400
mg/mL L-lysine solutions in various pH.
10401 FIG. 3 Appearances of the mixture of DCA-Na solutions and (a) 90 mg/mL,
(b) 180
mg/mL, (c) 300 mg/mL, (d) 450 mg/mL or (e) 600 mg/mL LA solutions.
10411 FIG. 4 Appearances of the mixture of DCA-Na solutions and (a) 90 mg/mL,
(b) 180
mg/mL, (c) 300 mg/mL, (d) 450 mg/mL or (e) 600 mg/mL LA in Lidocaine HC1
solutions.
10421 FIG. 5 Photos of fat tissues collected at both sides (L: left
side, R: right side) of the 2
pigs, wherein (a) and (b) are from the first pig, and (c) and (d) are from the
second pig.
10431 FIG. 6 Appearances of the mixture of DCA-Na solutions and (a) 200 mg/mL
or (b) 400
mg/mL L-lysine/DSP solutions in various pH.
10441 FIG. 7 Photos of fat tissues collected at both sides (L: left
side, R: right side) of the 3
pigs, wherein (a) and (b) are from the first pig, (c) and (d) are from the
second pig, and (e) and (f)
are from the third pig.
10451 FIG. 8 Appearances of the mixture of DCA-Na solutions and (a) 2.5 mg/mL,
(b) 5
mg/mL, (c) 10 mg/mL, (d) 20 mg/mL, (e) 40 mg/mL or (0 50 mg/mL L-histidine
solutions.
10461 FIG. 9 Appearances of the mixture of DCA-Na solutions and 500 mg/mL L-
arginine
solutions.
10471 FIG. 10 Appearances of the mixture of DCA-Na solutions and (a) 0.1%, (b)
0.2%, (c)
0.3%, (d) 0.4%, (e) 0.5%, or (f) 0.6% L-histidine solutions.
CA 03220724 2023- 11- 28
Detailed Description of the Invention
Definitions
10481 In the invention, the following defmitions are applicable:
10491 The articles "a" and "an" are used in this invention to refer to one or
more than one (i.e.,
to at least one) of the grammatical object of the article. By way of example,
"an element" means
one element or more than one element.
10501 The term "and/or" is used in this invention to mean either "and" or "or"
unless indicated
otherwise.
10511 The term "effective amount" means an amount of a composition according
to the
invention which, in the context of which it is administered or used, is
sufficient to achieve the
desired effect or result. An effective amount can be determined by methods
known to those of skill
in the art.
10521 A "subject" is a mammal, e.g., a human, mouse, rat, guinea pig, dog,
cat, horse, cow, pig,
or non-human primate, such as a monkey, chimpanzee, baboon or rhesus. Subject
of the invention
is preferably a human.
10531 A "pharmaceutically acceptable excipient" may be used herein, and refers
to a compound
that is useful in preparing a pharmaceutical composition that is generally
safe, non-toxic and
neither biologically nor otherwise undesirable, and includes excipients that
are acceptable for
veterinary use or human pharmaceutical use. A pharmaceutically acceptable
excipient as used in
the specification and claims includes both one and more than one such
excipient. Suitable
excipients include: solvents, such as sterile water or water for injection;
lubricating agents such as
talc, magnesium stearate; wetting agents; emulsifying and suspending agents;
tonicity agent, such
as sodium chloride; acid, such as hydrochloric acid; base, such as sodium
hydroxide; buffer, such
as dibasic sodium phosphate; and preserving agents such as methyl- and
propylhydroxy-benzoates
and benzyl alcohol.
10541 A "cytolytic compound" may also be a detergent or a lipolytic compound.
Suitable
cytolytic compounds include, but are not limited to phosphatidylcholine,
deoxycholic acid or a
salt thereof. Cytolytic compound of the invention is preferably deoxycholic
acid or a salt thereof,
more preferably DCA-Na.
10551 Aspirin (acetylsalicylic acid) is a nonsteroidal anti-inflammatory drug
(NSAID) used to
reduce pain, fever, or inflammations but also suppresses the normal
functioning of platelets. Its
soluble salt lysine aspirin (LA) can be administered intravenously or
intramuscularly. After
CA 03220724 2023- 11- 28
administration, lysine aspirin is converted into acetylsalicylic acid and
metabolized into salicylic
acid.
[056] Dexamethasone is a glucocorticosteroid similar to a natural hormone
produced by
adrenal glands. It relieves inflammation (swelling, heat, redness, and pain)
and is used to treat
certain forms of arthritis, severe allergies, asthma and certain types of
cancer. Dexamethasone
sodium phosphate (DSP) is its sodium phosphate salt form.
[057] Lidocaine (or lignocaine) is a local anesthetic of the amino amide type
which can
temporarily blocks transmission of nerve impulses. It typically begins working
within several
minutes and lasts for half an hour to three hours after administered.
Lidocaine mixtures may also
be applied directly to the skin or mucous membranes to numb the area.
Examples
[058] The present invention can be better understood according to the
following examples.
However, it would be easy for a person skilled in the art to understand that
the contents
described in the examples are merely intended to illustrate the present
invention rather than limit
the present invention described in detail in the claims. Unless otherwise
indicated, compositions
of the present invention can be prepared by using commercially available
materials and utilizing
general techniques and procedures known to those skilled in the art.
DCA-Na solutions
[059] DCA-No (99%, Acros Organics, Geel, Belgium), NaOH, Na2HPO4 (Sigma-
Aldrich, St.
Louis, MO, USA) and NaCl (Honeywell, Charlotte, NC, USA) were added to 80 mL
water for
injection and then made up to 100 mL solution. Benzyl alcohol (Alfa Aesar,
Ward Hill, MA,
USA) was then added to the solution and additional sodium
hydroxide/hydrochloric acid was
added to adjust the pH value. The amounts and concentrations of various
ingredients were as
shown in Tables 1 and 2 to prepare 5% and 1% solutions respectively. Solutions
were sterilized
by autoclave for 30 minutes.
TABLE 1
Ingredients Amount (mg) Concentration
(mg/mL)
Sodium deoxycholate (DCA-No) 5280 52.8
Dibasic sodium phosphate (Na2HPO4) 142 1.42
Sodium chloride (NaCl) 438 4.38
Benzyl alcohol 900 9 mg
Water for injection Up to 100 mL
5% solution: 52.8 mg/mL DCA-No (equivalent to 50 mg/mL DCA, 100 mL, pH 8.3)
CA 03220724 2023- 11- 28
TABLE 2
Ingredients Amount (mg) Concentration
(mg/mL)
Sodium deoxycholate (DCA-Na) 1056 10.56
Sodium hydroxide (NaOH) 40 0.4
Dibasic sodium phosphate (Na2HPO4) 142 1.42
Sodium chloride (NaCl) 438 4.38
Benzyl alcohol 900 9.00
Water for injection Up to 100 mL -
1% solution: 10.56 mg/mL DCA-Na solution (equivalent to 10 mg/mL DCA, 100 mL,
pH 8.3)
10601 In the following examples, DCA-Na solutions were mixed with other
components to
prepare an injectable composition. Unless otherwise stated, the requirements
for the final
concentration of DCA-Na in the obtained compositions were? 70% of initial
solutions (?36.96
mg/mL for 5% solution, > 7.39 mg/mL for 1% solution). The appearances after
mixing DCA-Na
with other components were observed after placing at 25, 37 and 42 C for 20,
30, 45, 60 and 120
minutes. 200 [EL of the mixtures were also added to 200 [IL 0.9% saline
respectively and their
appearances were also observed after placing at 37 C for 20, 30, 45, 60 and
120 minutes. Photos
were taken and showin in the figures.
Example 1. Compositions of DCA-Na and L-lysine
10611 To test if compositions of DCA-Na and L-lysine form gel after mixing,
DCA-Na
solutions were mixed with acidic L-lysine solutions (pH 5.0-5.2, Acros
Organics) according to
TABLE 3.
TABLE 3
Group 1 2 3 4
5
L-Lysine (g) 1.0 1.0 1.0 1.0
1.0
ddH20 (mL) 10.0 5.0 3.3 2.5
2.0
Final concentration of L-lysine (mg/mL) 100 200 300 400
500
Group 1-1 1-2 1-3 1-4
1-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
100 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-lysine (mg/mL) 4.76 9.09 16.67
23.07 28.57
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Group 1-6 1-7 1-8 1-9
1-10
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
100 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80 8.12
7.54
Final concentration of L-lysine (mg/mL) 4.76 9.09 16.67
23.07 28.57
Group 2-1 2-2 2-3 2-4
2-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
200 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-lysine (mg/mL) 9.52 18.18 33.33
46.15 57.14
Group 2-6 2-7 2-8 2-9
2-10
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
200 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80 8.12
7.54
Final concentration of L-lysine (mg/mL) 9.52 18.18 33.33
46.15 57.14
Group 3-1 3-2 3-3 3-4
3-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
300 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-lysine (mg/mL) 14.29 27.27 50.00
69.23 85.71
Group 3-6 3-7 3-8 3-9
3-10
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
300 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80 8.12
7.54
Final concentration of L-lysine (mg/mL) 14.29 27.27 50.00
69.23 85.71
Group 4-1 4-2 4-3 4-4
4-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
400 mg/mL L-lysine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-lysine (mg/mL) 18.87 36.36 66.67
92.31 114.29
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Group 4-6 4-7 4-8 4-
9 4-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
400 mg/mL L-lysine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-lysine (mg/mL) 18.87 36.36 66.67
92.31 114.29
Group 5-1 5-2 5-3 5-
4 5-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
500 mg/mL L-lysine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-lysine (mg/mL) 23.81 45.45 83.33
115.38 142.86
Group 5-6 5-7 5-8 5-
9 5-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
500 mg/mL L-lysine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-lysine (mg/mL) 23.81 45.45 83.33
115.38 142.86
[062] FIG. 1 showed that all groups formed transparent solution when lysine
solutions were
added to DCA-Na solutions. Mixtures of DCA-Na and lysine with higher
concentration of lysine
(FIG. lc-e) started to form gel (remained at the bottom of the bottle after
inverted) around 30
minutes when placed at 25 C, while mixtures of DCA-Na and lysine placed at 42
C did not form
gel at all tested lysine concentration in 5% DCA-Na and lysine concentration
lower than 140
mg/mL in 1% DCA-Na. Mixtures of 5% DCA-Na and lysine added to 0.9% saline
formed gel
around 60 minutes at lysine concentration >83 mg/mL and around 30 minutes at
lysine
concentration >85 mg/mL (FIG. lc-e). Mixtures of 1% DCA-Na and lysine added to
0.9% saline
formed gel around 60 minutes at lysine concentration >45 mg/mL; around 30
minutes at lysine
concentration >69 mg/mL and around 20 minutes at lysine concentration >85
mg/mL (FIG. lc-e).
These results showed that higher concentration of LA formed gel in shorter
time. Therefore,
mixtures of DCA-Na and lysine are suggested to be used as soon as possible
after mixing.
[063] In Example 1, the compositions added with 0.9% saline can form gel when
the final
concentration of DCA-Na was 7.54-44.00 mg/mL, and the final concentration of L-
lysine was
45.45-142.86 mg/mL.
CA 03220724 2023- 11- 28
[064] To test for the optimal pH values for DCA-Na solutions and L-lysine
solutions that form
gel after mixing, DCA-Na solutions were mixed with L-lysine with various pH
according to
TABLE 4.
TABLE 4
Group 1 2
L-lysine (g) 0.6 1.2
ddH20 (mL) 3.0 3.0
Final concentration of lysine (mg/mL) 200 400
Initial pH 10.3-10.4
Group 1-1 1-2 1-3 1-4 1-5 1-
6 1-7
5% DCA-Na solution (mL) 1.00
200 mg/mL L-lysine (mL) 0.40
Final concentration of
37.7
DCA-Na (mg/mL)
Final concentration of
57.14
L-lysine (mg/mL)
pH of L-lysine solution (
4.0 5.0 6.0 7.0 8.0
9.0 10.0
0.02)
Group 1-8 1-9 1-10 1-11 1-12 1-
13 1-14
1% DCA-Na solution (mL) 1.00
200 mg/mL L-lysine (mL) 0.40
Final concentration of
7.54
DCA-Na (mg/mL)
Final concentration of
57.14
L-lysine (mg/mL)
pH of L-lysine solution (
4.0 5.0 6.0 7.0 8.0
9.0 10.0
0.02)
* DCA-Na solutions mixed with pH 3.0 L-lysine solution (200 mg/mL) formed
precipitation.
Group 2-1 2-2 2-3 2-4 2-5
2-6
5% DCA-Na solution (mL) 1.00
200 mg/mL L-lysine (mL) 0.40
Final concentration of
37.7
DCA-Na (mg/mL)
Final concentration of
114.29
L-lysine (mg/mL)
pH of L-lysine solution (+
5.0 6.0 7.0 8.0 9.0
10.0
0.02)
Group 2-7 2-8 2-9 2-10 2-11
2-12
CA 03220724 2023- 11- 28
1% DCA-Na solution (mL) 1.00
200 mg/mL L-lysine (mL) 0.40
Final concentration of
7.54
DCA-Na (mg/mL)
Final concentration of
114.29
L-lysine (mg/mL)
pH of L-lysine solution (
5.0 6.0 7.0 8.0 9.0 10.0
0.02)
* DCA-Na solutions mixed with pH 4.0 L-lysine solution (400 mg/mL) formed
precipitation.
TABLE 5
pH of L-lysine solution 4.0 5.0 6.0 7.0 8.0 9.0
10.0
Group 1-1 1-2 1-3 1-4 1-5 1-6
1-7
pH of L-lysine solution +
7.21 7.35 7.38 7.58 8.09 9.05 9.97
5% DCA-Na solution
Group 1-8 1-9 1-10 1-11 1-12 1-13
1-14
pH of L-lysine solution +
6.71 7.02 7.09 7.42 8.05 8.98 9.92
1% DCA-Na solution
pH of L-lysine solution 5.0 6.0 7.0 8.0 9.0
10.0
Group 2-1 2-2 2-3 2-4 2-5
2-6
pH of L-lysine solution +
7.45 7.55 7.70 8.26 9.07 9.92
5% DCA-Na solution
Group 2-7 2-8 2-9 2-10 2-11
2-12
pH of L-lysine solution +
6.96 7.22 7.50 8.23 9.04 9.89
1% DCA-Na solution
10651 FIG. 2 showed that all groups formed transparent solution when lysine
solutions were
added to DCA-Na solutions. The pH value of mixed solutions ranged from 7.21-
9.97 and
6.71-9.92 in 5% and 1% DCA-Na solution mixed with 200 mg/mL L-lysine solution
at pH
ranged from 4.0 to 10.0; 7.45-9.92 and 6.96-9.89 in 5% and 1% DCA-Na solution
mixed with
400 mg/mL L-lysine solution at pH ranged from 5.0 to 10.0 (TABLE 5). At 200
mg/mL L-lysine
test, 5% DCA-Na and L-lysine added to 0.9% saline formed gel around 60 minutes
at pH 4.0;
mixtures of 1% DCA-Na and L-lysine added to 0.9% saline formed gel around 30
minutes at pH
4.0, around 45 minutes at pH 5.0 (FIG. 2a). At 400 mg/mL L-lysine test,
mixtures of 5%
DCA-Na and L-lysine added to 0.9% saline formed gel around 45 minutes at pH
5.0 and 6.0,
around 60 minutes at pH 7.0; mixtures of 1% DCA-Na and L-lysine added to 0.9%
saline formed
gel around 30 minutes at pH 5.0, around 45 minutes at pH 6.0 (FIG. 2b).
Therefore, suitable pH
CA 03220724 2023- 11- 28
for L-lysine solution before mixing is <8.0, preferably 5.0-7.0, more
preferably around pH
5.0-6Ø Lower pH value is suggested for lower concentration of L-lysine.
10661 The compositions added with 0.9% saline can form gel when the final pH
of the
composition was 7.02-7.70.
Example 2. Compositions of DCA-Na and Lysine Aspirin
10671 To test if mixing DCA-Na solutions with lysine-containing NSAID can form
gel,
DCA-Na solutions were mixed with LA (Lyacety, 0.9 g/bottle, equivalent to 0.5
g aspirin, China
Chemical & Pharmaceutical Co., Ltd., Taipei City, China) according to TABLE 6.
TABLE 6
Group 1 2 3 4
5
Lysine aspirin (g) 0.9 0.9 0.9 0.9
0.9
ddH20 (mL) 10.0 5.0 3.0 2.0
1.5
Final concentration of LA (mg/mL) 90 180 300 450
600
Group 1-1 1-2 1-3 1-4
1-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
90 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of LA (mg/mL) 4.29 8.18 15.00
20.77 25.71
Group 1-6 1-7 1-8 1-9
1-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
90 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of LA (mg/mL) 4.29 8.18 15.00
20.77 25.71
Group 2-1 2-2 2-3 2-4
2-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
180 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of LA (mg/mL) 8.57 16.36 30.00
41.54 51.43
Group 2-6 2-7 2-8 2-9
2-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
CA 03220724 2023- 11- 28
180 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of LA (mg/mL) 8.57 16.36 30.00
41.54 51.43
Group 3-1 3-2 3-3 3-
4 3-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
300 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of LA (mg/mL) 14.29 27.27 50.00
69.23 85.71
Group 3-6 3-7 3-8 3-
9 3-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
300 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of LA (mg/mL) 14.29 27.27 50.00
69.23 85.71
Group 4-1 4-2 4-3 4-
4 4-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
450 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of LA (mg/mL) 21.43 40.91 75.00
103.85 128.57
Group 4-6 4-7 4-8 4-
9 4-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
450 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of LA (mg/mL) 21.43 40.91 75.00
103.85 128.57
Group 5-1 5-2 5-3 5-
4 5-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
600 mg/mL LA (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of LA (mg/mL) 28.57 54.55 100.00
138.46 171.43
Group 5-6 5-7 5-8 5-
9 5-10
CA 03220724 2023- 11- 28
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00
1.00
600 mg/mL LA (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80 8.12
7.54
Final concentration of LA (mg/mL) 28.57 54.55 100.00
138.46 171.43
10681 FIG. 3 showed that all groups formed transparent solution when LA
solutions were
added to DCA-Na solutions. Mixtures of DCA-Na and LA with higher concentration
of LA
started to form gel around 20 minutes when placed at 25 C (FIG. 3d, e), while
mixtures placed at
37 or 42 C took longer time to form gel but formed suspension (or
precipitation) within a short
period of time (FIG. 3b-e). Mixtures added to 0.9% saline formed gel around 60
minutes at LA
concentration >50 mg/mL; around 30 minutes at LA concentration >69 mg/mL (FIG.
3b-e).
Higher concentration of LA formed gel in shorter time. Therefore, mixtures of
DCA-Na and LA
are suggested to be used as soon as possible after mixing.
10691 In Example 2, the compositions added with 0.9% saline can form gel when
the final
concentration of DCA-Na was 7.54-48.00 mg/mL, even up to 50.29 mg/mL; and the
final
concentration of LA was 25.71-171.43 mg/mL, wherein the final concentrations
of lysine and
aspirin were about 11.40-76.81 mg/mL and 14.31-94.62 mg/mL, respectively.
Example 3. Compositions of DCA-Na and Lysine Aspirin with Lidocaine HC1
10701 To test if DCA-Na solutions and LA dissolved in local anesthetic
lidocaine HC1 form
gel after mixing, DCA-Na solutions were mixed with LA in lidocaine HC1 (5
mL/bottle, Lita
Pharmacy CO., Ltd., Taichung City, China) according to TABLE 7.
TABLE 7
Group 1 2 3 4
5
Lysine aspirin (g) 0.9 0.9 0.9 0.9
0.9
Lidocaine HC1 (mL) 10.0 5.0 3.0 2.0
1.5
Final concentration of LA (mg/mL) 90 180 300 450
600
Group 1-1 1-2 1-3 1-4
1-5
5% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
90 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 44.90 44.00 40.6
37.71 36.97
Final concentration of LA (mg/mL) 13.47 15.00 20.77
25.71 26.97
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
CA 03220724 2023- 11- 28
Group 1-6 1-7 1-8 1-9
1-10
1% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
90 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 8.98 8.80 8.12 7.54
7.39
Final concentration of LA (mg/mL) 13.47 15.00 20.77
25.71 26.97
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 2-1 2-2 2-3 2-4
2-5
5% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
180 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 44.90 44.00 40.6
37.71 36.97
Final concentration of LA (mg/mL) 26.94 30.00 41.54
51.43 53.95
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 2-6 2-7 2-8 2-9
2-10
1% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
180 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 8.98 8.80 8.12 7.54
7.39
Final concentration of LA (mg/mL) 26.94 30.00 41.54
51.43 53.95
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 3-1 3-2 3-3 3-4
3-5
5% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
300 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 44.90 44.00 40.6
37.71 36.97
Final concentration of LA (mg/mL) 44.90 50.00 69.23
85.71 89.92
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 3-6 3-7 3-8 3-9
3-10
1% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
300 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 8.98 8.80 8.12 7.54
7.39
Final concentration of LA (mg/mL) 44.90 50.00 69.23
85.71 89.92
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
CA 03220724 2023- 11- 28
Group 4-1 4-2 4-3 4-4
4-5
5% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
450 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 44.90 44.00 40.6
37.71 36.97
Final concentration of LA (mg/mL) 67.35 75.00 103.85
128.57 134.87
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 4-6 4-7 4-8 4-9
4-10
1% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
450 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 8.98 8.80 8.12 7.54
7.39
Final concentration of LA (mg/mL) 67.35 75.00 103.85
128.57 134.87
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 5-1 5-2 5-3 5-4
5-5
5% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
600 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 44.90 44.00 40.6
37.71 36.97
Final concentration of LA (mg/mL) 89.80 100.00 138.46
171.43 179.83
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
Group 5-6 5-7 5-8 5-9
5-10
1% DCA-Na solution (mL) 1.000 1.000 1.000
1.000 1.000
600 mg/mL LA/lidocaine (mL) 0.176 0.200 0.300
0.400 0.428
Final concentration of DCA-Na (mg/mL) 8.98 8.80 8.12 7.54
7.39
Final concentration of LA (mg/mL) 89.80 100.00 138.46
171.43 179.83
Final concentration of lidocaine (mg/mL) 2.99 3.33 4.62 5.72
5.99
10711 FIG. 4 showed that all groups formed transparent solution when LA in
lidocaine HCl
solutions were added to DCA-Na solutions. Mixtures of DCA-Na and LA in
lidocaine HC1 with
high concentration of LA started to form gel around 30 minutes when placed at
25 C (FIG. 4e),
while mixtures placed at 37 or 42 C took longer time to form gel but formed
suspension or
precipitation within a short period of time (FIG. 4a-e). For 5% DCA-Na
solution, mixtures of
DCA-Na and LA in lidocaine HC1 added to 0.9% saline formed gel around 60
minutes at LA
concentration >70 mg/mL; around 45 minutes at LA concentration >134 mg/mL;
around 30
CA 03220724 2023- 11- 28
minutes at LA concentration >170 mg/mL. (FIG. 4c-e). Concentration of
lidocaine HC1 mixing
with 5% DCA-Na solution was tolerable up to 6 mg/mL. For 1% DCA-Na solution,
mixtures of
DCA-Na and LA + lidocaine HC1 added to 0.9% saline formed gel around 120
minutes at LA
concentration >40 mg/mL; around 60 minutes at LA concentration >67 mg/mL;
around 45
minutes at LA concentration >85 mg/mL (FIG. 4b-e). Suitable concentration of
lidocaine HC1
mixing with 1% DCA-Na solution was around 3 mg/mL. These results showed that
high
concentration of lidocaine added to low concentration of DCA-Na precipitated
easily.
10721 In Example 3, the compositions added with 0.9% saline can form gel when
the final
concentration of DCA-Na was 8.12-44.90 mg/mL; the final concentration of LA
was
41.54-179.83 mg/mL, wherein the final concentrations of lysine and aspirin
were about
18.61-80.61 mg/mL and 22.93-99.22 mg/mL, respectively; and the final
concentration of
lidocaine was 2.99-6.99 mg/mL.
Example 4. Effects of DCA-Na and Lysine Aspirin with Lidocaine HC1 in Porcine
Tissue
10731 2 male SPF Landrace pigs aging around 5-6 months were anesthetized via
intramuscular
injection of 0.04 mg/kg Atropine. After 10-15 minutes, 6 mg/kg Zoletil 50 and
2.2 mg/kg
Rompun were injected intramuscularly.1.5 mL lidocaine HC1 were added to LA and
mixed until
dissolved. 0.35 mL lidocaine HC1/LA solution were added to 2 mL 1% or 5% DCA-
Na solution
and mixed until dissolved. Pigs were injected with 0.9% saline, 1% or 5% DCA-
Na solutions
with or without lidocaine HC1/LA at different time points according to TABLE
8. Area for each
injection site is 16 cm2 and compositions were injected at a depth of 1.0 cm
at the center of each
site. 55 sites were injected at each side of the pigs (Total 110 sites/pig).
After sacrificed (day 0),
fat tissue samples were collected and cut in half from the center. Photos of
sections were
recorded and shown in FIG. 5.
TABLE 8
Amount of single
Group Composition Time points for injection
injection (IL)
1 Blank - -
2 0.9% Saline -3 hour 200
3 0.9% Saline -3 hour 400
4-1 4-2 4-3 4-4 4-5 4-6
4-1-4-6 1% DCA-Na solution 200
-28 day -21 day -14 day -7 day -2 day -3 hour
5-1 5-2 5-3 5-4 5-5 5-6
5-1-5-6 1% DCA-Na solution 400
-28 day -21 day -14 day -7 day -2 day -3 hour
6-1-6-6 5% DCA-Na solution 6-1 6-2 6-3 6-4 6-5 6-6
200
CA 03220724 2023- 11- 28
-28 day -21 day -14 day -7 day -2 day -3 hour
7-1 7-2 7-3 7-4 7-5 7-6
7-1-7-6 5% DCA-Na solution 400
-28 day -21 day -14 day -7 day -2 day -3 hour
1% DCA-Na solution + 8_1 8_2 8_3 8_4 8_5 8_6
8-1-8-6 200
LA in Lidocaine HC1 -28 day -21 day -14 day -7 day -2 day -3 hour
9 9 6 1% DCA-Na solution + 9-1 9-2 9-
3 9-4 9-5 9-6 400
¨
-1 - LA in Lidocaine HC1 -28 day -21 day -14 day -7 day -2 day
-3 hour
5% DCA-Na solution +
10-1¨ 10-1 10-2 10-3 10-4 10-5 ..
10-6
10-6 LA in Lidocaine HC1 -28 day -21 day -14 day -7 day -2 day -
3 hour 200
5% 11-1 DCA-Na solution + 11-1 11-2 11-3 11-4 11-5
11-6
¨
11-6 LA in Lidocaine HCl -28 day -21 day -14 day -7 day -2 day -
3 hour 400
1% DCA-Na solution +
12 LA in Lidocaine HCl -28 day, -21 day, -14 day, -7 day 200
1% DCA-Na solution +
13 LA in Lidocaine HC1 -28 day, -21 day, -14 day, -7 day 400
5% DCA-Na solution +
14 LA in Lidocaine HC1 -28 day, -21 day, -14 day, -7 day 200
5% DCA-Na solution +
15 LA in Lidocaine HC1 -28 day, -21 day, -14 day, -7 day 400
10741 As shown in FIG. 5, 7 days after injection, sites injected with DCA-Na
solution alone
were slightly harder and much swollen than sites injected with DCA-Na
solutions with lidocaine
HC1/LA. FIG. 5 showed that cytolysis occurs at injected site if DCA-Na
solutions were injected
alone (Group 4-7). On the other hand, cytolysis occurs at the bottom of fat
tissue if DCA-Na
solutions were injected along with lidocaine HC1/LA (Group 8-15). This might
suggest that
DCA-Na solutions alone tend to diffuse in fat tissue, however, mixing DCA-Na
solutions with
lidocaine HC1/LA formed gel that might deposit and diffuse at the bottom of
the fat tissue, which
coincide with less hardness being palpated. Cytolysis and/or inflammation at
sites injected with
DCA-Na solution alone were observable for at least 21-28 days but were less
observable after 21
days at sites injected with DCA-Na solutions with lidocaine HC1/LA.
10751 Compositions of DCA-Na and Lysine Aspirin with Lidocaine HC1 can
effectively
reduce fat, with less adverse effects, such as inflammation.
Example 5. Compositions of DCA-Na and L-lysine with DSP
10761 To test if mixing DCA-Na solutions with lysine and another anti-
inflammatory drug,
DSP (Tai Yu Chemical & Pharmaceutical Co., Ltd., Hsinchu County, China), can
form gel and
its optimal pH value for forming gel, DCA-Na solutions were mixed with L-
lysine/DSP of
CA 03220724 2023- 11- 28
different pH values according to TABLE 9. Requirement: Final concentration of
DSP: 1
mg/mL.
TABLE 9
Group 1 2
L-lysine (g) 0.6
1.2
DSP (mg) 13.0
13.0
ddH20 (mL) 3.0
3.0
Final concentration of L-lysine (mg/mL) 200
400
Final concentration of DSP (mg/mL) 4.33
4.33
Group 1-1 1-2 1-3
1-4
5% DCA-Na solution (mL) 1.00
L-lysine (200 mg/mL)/DSP solution (mL) 0.30
Final concentration of DCA-Na (mg/mL) 40.615
Final concentration of L-lysine (mg/mL) 46.154
Final concentration of DSP (mg/mL) 0.999
pH of L-lysine solution (+ 0.10) 4.0 5.0 6.0
7.0
Group 1-5 1-6 1-7
1-8
1% DCA-Na solution (mL) 1.00
L-lysine (200 mg/mL)/DSP solution (mL) 0.30
Final concentration of DCA-Na (mg/mL) 8.123
Final concentration of L-lysine (mg/mL) 46.154
Final concentration of DSP (mg/mL) 0.999
pH of L-lysine solution (+ 0.02) 4.0 5.0 6.0
7.0
Group 2-1 2-2 2-3
2-4
5% DCA-Na solution (mL) 1.00
L-lysine (400 mg/mL)/DSP solution (mL) 0.30
Final concentration of DCA-Na (mg/mL) 40.615
Final concentration of L-lysine (mg/mL) 92.308
Final concentration of DSP (mg/mL) 0.999
pH of L-lysine solution (+ 0.10) 4.0 5.0 6.0
7.0
Group 2-5 2-6 2-7
2-8
CA 03220724 2023- 11- 28
1% DCA-Na solution (mL) 1.00
L-lysine (200 mg/mL)/DSP solution (mL) 0.30
Final concentration of DCA-Na (mg/mL) 8.123
Final concentration of L-lysine (mg/mL) 92.308
Final concentration of DSP (mg/mL) 0.999
pH of L-lysine solution ( 0.02) 4.0 5.0 6.0
7.0
TABLE 10
pH of L-lysine solution 4.0 5.0 6.0
7.0
Group 1-1 1-2 1-3
1-4
pH of L-lysine/DSP solution + 6.87 7.15 7.21
7.43
5% DCA-Na solution
Group 1-5 1-6 1-7
1-8
pH of L-lysine/DSP solution + 6.48 6.81 6.96
7.28
1% DCA-Na solution
pH of L-lysine solution 4.0 5.0 6.0
7.0
Group 24 2-2 2-3
2-4
pH of L-lysine/DSP solution + 7.11 7.27 7.38
7.54
5% DCA-Na solution
Group 2-5 2-6 2-7
2-8
pH of L-lysine/DSP solution + 6.75 7.01 7.17
7.34
1% DCA-Na solution
10771 FIG. 6 showed that all groups formed transparent solution when L-
lysine/DSP solutions
were added to DCA-Na solutions. The pH value of mixed solutions ranged from
6.87-7.43 and
6.48-7.28 in 5% and 1% DCA-Na solution mixed with 200 mg/mL L-lysine solution
at pH
ranged from 4.0 to 7.0; 7.11-7.54 and 6.75-7.34 in 5% and 1% DCA-Na solution
mixed with 400
mg/mL L-lysine solution at pH ranged from 4.0 to 7.0 (TABLE 10). At 200 mg/mL
L-lysine test,
mixtures of 5% DCA-Na and L-lysine/DSP added to 0.9% saline formed gel around
45 minutes
at pH 4.0; mixtures of 1% DCA-Na and L-lysine/DSP added to 0.9% saline formed
gel around
20 minutes at pH 4.0 (FIG. 6a). At 400 mg/mL L-lysine test, mixtures of 5% DCA-
Na and
L-lysine/DSP added to 0.9% saline formed gel around 30 minutes at pH 4.0 and
5.0, around 45
minutes at pH 6.0; mixtures of 1% DCA-Na and L-lysine/DSP added to 0.9% saline
formed gel
around 20 minutes at pH 4.0, around 30 minutes at pH 5.0 and 6.0 (FIG. 6b).
This suggested that
DCA-Na solutions can form gel after mixing with L-lysine DSP solutions and the
time were
CA 03220724 2023- 11- 28
shortened if concentration of L-lysine increased. Suitable pH for L-lysine/DSP
solution is around
pH 4.0-6Ø
10781 In Example 5, the compositions added with 0.9% saline can form gel when
the final
concentration of DCA-Na was 8.123 or 40.615 mg/mL; the final concentration of
lysine was
46.154 or 92.308 mg/mL; and the final concentration of DSP was 0.999 mg/mL.
The
compositions added with 0.9% saline can form gel when the final pH of the
composition was
6.48-7.38.
Example 6. Effects of DCA-Na and Lysine with DSP in Porcine Tissue
10791 3 male pigs weighting at least 100 kg were anesthetized via
intramuscular injection of
0.02 mg/kg Atropine and along with inhalation of 3 % Isoflurane and 30-70%
nitrous oxide
(N20) mixed with oxygen (02). 0.5 mL L-lysine/DSP solution (pH 6.0) were added
to 1 mL 1%
or 5% DCA-Na solution and mixed until dissolved. Pigs were injected with 0.9%
saline, 1% or 5%
DCA-Na solutions with L-lysine/DSP solutions at different time points
according to TABLE 11.
Area for each injection site is 9 cm2 and compositions were injected at a
depth of 0.5 cm at the
center of each site. 54 sites were injected at each side of the pigs (Total
108 sites/pig). At day 0,
animals were anesthetized via intramuscular injection of 0.02 mg/kg Atropine
and 6 mg/kg
Zoletil 50. Fat tissue samples were collected and cut in half from the center.
Photos of tissues
sections were recorded and shown in FIG. 7.
TABLE 11
L-lysine L-lysine/ DSP L-lysine/ DSP L-lysine/
DSP L-lysine/ DSP
solution 0.1% solution 0.2 % solution 0.4%
solution 0.5% solution
L-lysine (g) 1.5 1.5 1.5 1.5
1.5
DSP (mg) 0.0 3.0 6.0 12.0
24.0
ddH20 (mL) 3.0 3.0 3.0 3.0
3.0
Final concentration
500.0 500.0 500.0 500.0
500.0
of L-lysine (mg/mL)
Final concentration
0.0 1.0 2.0 4.0 5.0
of DSP (mg/mL)
Amount of
Group Composition Time points for injection
single injection
( L)
0 Blank -
1-1 1-2 1-3 1-4 1-5 1-6
1-1 - 1-6 0.9% saline
200
-28 day -21 day -14 day -7 day -2 day -3 hour
CA 03220724 2023- 11- 28
2-1 2-2 2-3 2-4 2-5 2-6
2-1 2-6
1% DCA-Na solution + Lysine 200
¨
HC1 -28 day -21 day -14 day -7 day -2 day -
3 hour
3-1 3-2 3-3 3-4 3-5 3-6
3-1 3-6
1% DCA-Na solution + Lysine 400
¨
HC1 -28 day -21 day -14 day -7 day -2 day -
3 hour
4-1 4-2 4-3 4-4 4-5 4-6
4-1 4-6
5% DCA-Na solution + Lysine 200
¨
HC1 -28 day -21 day -14 day -7 day -2 day -
3 hour
. 5-1 5-2 5-3 5-4 5-5 5-6
5% 5-1 5-6
DCA-Na solution + Lysine 400
¨
HC1 -28 day -21 day -14 day -7 day -2 day -
3 hour
6-6 6-1 6-2 6-3 6-4 6-5 6-6
6-1
5% DCA-Na solution + Lysine 200
¨
HC1/DSP 0.1% solution -28 day -21 day -14 day -7 day -2 day -
3 hour
7-6 7-1 7-2 7-3 7-4 7-5 7-6
7-1
5% DCA-Na solution + Lysine 200
¨
HC1/DSP 0.2 % solution -28 day -21 day -14 day -7 day -2 day -3 hour
. 8-1 8-2 8-3 8-4 8-5 8-6
5% 8-6 8-1 DCA-Na solution + Lysine 200
¨
HC1/DSP 0.4 % solution -28 day -21 day -14 day -7 day -2 day -3 hour
9-6 9-1 9-2 9-3 9-4 9-5 9-6
9-1
5% DCA-Na solution + Lysine 200
¨
HC1/DSP 0.8 % solution -28 day -21 day -14 day -7 day -2 day -3 hour
10801 FIG. 7 showed that cytolysis occured at injected site when DCA-Na
solutions were
injected along with lysine or lysine/DSP at shallower depth. Increasing
concentration or volume
of DCA-Na resulted in stronger cytolytic reaction or inflammation as larger
area of redness were
observed (Groups 2-5). The cytolytic reaction or inflammation were much
relieved after 7-14
days of injection, as less redness were observed. Increasing concentration of
DSP also reduce the
degree and area redness at injected site (Groups 6-9), suggesting that the
additional of
anti-inflammatory DSP could effectively reduce inflammation at injected site.
10811 Compositions of DCA-Na and Lysine with DSP can effectively reduce fat,
with less
adverse effects, such as inflammation and redness.
Example 7. Compositions of DCA-Na and Basic Amino Acids
10821 To test if DCA-Na solutions and other basic, cationic amino acids form
gel after mixing,
DCA-Na solutions were mixed with acidic L-histidine (pH 5.0-5.2, Sigma-
Aldrich) or L-arginine
(pH 5.0-5.2, Sigma-Aldrich) solutions according to TABLES 12 and 13,
respectively.
Example 7.1. Compositions of DCA-Na and L-histidine
TABLE 12
Group 1 2 3 4 5
6
L-Histidine (g) 0.025 0.05 0.10 0.20
0.40 0.50
CA 03220724 2023- 11- 28
ddH20 (mL) 10.0 10.0 10.0
10.0 10.0 10.0
Final concentration of L-histidine (mg/mL) 2.5 5 10 20 40
50
Group 1-1 1-2 1-3 1-
4 1-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
2.5 mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00
44.00 40.62 37.7
Final concentration of L-histidine (mg/mL) 0.12 0.23 0.42
0.58 0.71
Group 1-6 1-7 1-8 1-
9 1-10
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
2.5 mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-histidine (mg/mL) 0.12 0.23 0.42
0.58 0.71
Group 2-1 2-2 2-3 2-
4 2-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
5 mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-histidine (mg/mL) 0.24 0.45 0.83
1.15 1.43
Group 2-6 2-7 2-8 2-
9 2-10
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
5 mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-histidine (mg/mL) 0.24 0.45 0.83
1.15 1.43
Group 3-1 3-2 3-3 3-
4 3-5
5% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
10 mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-histidine (mg/mL) 0.48 0.91 1.67
2.31 2.86
Group 3-6 3-7 3-8 3-
9 3-10
1% DCA-Na solution (mL) 1.00 1.00 1.00 1.00 1.00
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mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-histidine (mg/mL) 0.48 0.91 1.67
2.31 2.86
Group 4-1 4-2 4-3 4-
4 4-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
mg/mL L-histidine (mL) 0.05 0.10 0.20 0.30
0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-histidine (mg/mL) 0.95 1.82 3.33
4.62 5.71
Group 4-6 4-7 4-8 4-
9 4-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
20 mg/mL L-histidine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-histidine (mg/mL) 0.95 1.82 3.33
4.62 5.71
Group 5-1 5-2 5-3 5-
4 5-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
40 mg/mL L-histidine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-histidine (mg/mL) 1.90 3.64 6.67
9.23 11.43
Group 5-6 5-7 5-8 5-
9 5-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
40 mg/mL L-histidine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of L-histidine (mg/mL) 1.90 3.64 6.67
9.23 11.43
Group 6-1 6-2 6-3 6-
4 6-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
50 mg/mL L-histidine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of L-histidine (mg/mL) 2.38 4.55 8.33
11.54 14.29
Group 6-6 6-7 6-8 6-
9 6-10
CA 03220724 2023- 11- 28
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
50 mg/mL L-histidine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80 8.12
7.54
Final concentration of L-histidine (mg/mL) 2.38 4.55 8.33
11.54 14.29
10831 L-histidine solutions with concentration higher than 2.86 mg/mL
precipitated after
added to 1% DCA-Na solution (FIG. 8c-f); L-histidine solutions with
concentration higher than
11.43 mg/mL precipitated after added to 5% DCA-Na solution (FIG. 8e-f).
Mixtures of DCA-Na
and L-histidine with higher concentration of L-histidine started to form gel
around 20 minutes
when placed at 25 C, while mixtures placed at 37 or 42 C formed suspensions
(or precipitations)
within a short period of time (FIG. 8b-f). Mixtures of 1% DCA-Na and L-
histidine added to 0.9%
saline formed gel around 20 minutes at L-histidine concentration 21.43 mg/mL
(FIG. 8b-e).
Mixtures of 5% DCA-Na and L-histidine added to 0.9% saline formed gel around
20 minutes at
L-histidine concentration 22.86 mg/mL (FIG. 8c-e).
10841 In Example 7.1, the compositions added with 0.9% saline can form gel
when the final
concentration of DCA-Na was 7.54-48.00 mg/mL, and the final concentration of L-
histidine was
1.43-11.43 mg/mL.
Example 7.2. Compositions of DCA-Na and L-arginine
TABLE 13
Group 1
L-arginine(g) 5.0
ddH20 (mL) 10.0
Final concentration of L-arginine (mg/mL) 500
*600 mg/mL L-arginine do not dissolve in ddH20.
Group 1-1 1-2 1-3 1-4
1-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
500 mg/mL L-arginine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00 40.62
37.7
Final concentration of L-arginine (mg/mL) 23.81 45.45 83.33 115.38
142.86
Group 1-6 1-7 1-8 1-9
1-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
500 mg/mL L-arginine (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 .. 9.60 .. 8.80 .. 8.12 ..
7.54
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Final concentration of L-arginine (mg/mL) 23.81 45.45 83.33
115.38 142.86
10851 FIG. 9 showed that all groups formed transparent solution when 500 mg/mL
L-arginine
solutions were added to DCA-Na solutions. However, only group 1-10 formed gel
around 60
minutes when placed at 25 C and after added to 0.9% saline. All mixtures of
DCA-Na and
L-arginine placed at 37 or 42 C did not formed gel in all tested time.
10861 In Example 7.2, the compositions added with 0.9% saline can form gel
when the final
concentration of DCA-Na was 7.54 or 8.12 mg/mL, and the final concentration of
L-arginine
was 115.38 or 142.86 mg/mL.
10871 These results revealed that although L-lysine, L-histidine and L-
arginine belong to basic
amino acids, the concentrations required to form gel were different. For
instance, only high
concentration of L-arginine and low concentration of DCA-Na formed gel and
took longer time
compared to L-lysine and L-histidine. On the other hand, low concentration of
L-histidine was
sufficient to form gel. In terms of forming gel compositions with DCA-Na,
lysine may be the
best, followed by histidine and arginine the worst.
Example 8. Compositions of DCA-Na and Organic Acid
10881 We have shown that pH value of solutions mixed with DCA-Na solutions
affect the
ability to form gel. To test if DCA-Na solutions and organic acid form gel
after mixing, DCA-Na
solutions were mixed with diluted acetic acid (Scharlau, Barcelona, Spain)
according to TABLE
14.
TABLE 14
Group 1 2 3 4 5
6
Acetic acid (mL) 0.01 0.02 0.03
0.04 0.05 0.06
ddH20 (mL) 10.0 10.0 10.0
10.0 10.0 10.0
Final concentration of acetic acid (%) 0.1 0.2 0.3 0.4
0.5 0.6
Group 1-1 1-2 1-3 1-4
1-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.1% acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of acetic acid (x 10-3 %) 4.76 9.09 16.67
23.08 28.57
Group 1-6 1-7 1-8 1-9
1-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.1% acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
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Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of acetic acid (x 10-3 %) 4.76 9.09 16.67
23.08 28.57
Group 2-1 2-2 2-3 2-
4 2-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.2 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of acetic acid (x 10-3 %) 9.52 18.18 33.33
46.15 57.14
Group 2-6 2-7 2-8 2-
9 2-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.2 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of acetic acid (x 10-3 %) 9.52 18.18 33.33
46.15 57.14
Group 3-1 3-2 3-3 3-
4 3-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.3 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of acetic acid (x 10-3 %) 14.29 27.27 50.00
69.23 85.71
Group 3-6 3-7 3-8 3-
9 3-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.3 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of acetic acid (x 10-3 %) 14.29 27.27 50.00
69.23 85.71
Group 4-1 4-2 4-3 4-
4 4-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.4 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of acetic acid (x 10-3 %) 19.05 36.36 66.67
92.30 114.29
Group 4-6 4-7 4-8 4-
9 4-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
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0.4 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of acetic acid (x 10-3 %) 19.05 36.36 66.67
92.30 114.29
Group 5-1 5-2 5-3 5-
4 5-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.5% acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of acetic acid (x 10-3 %) 23.81 45.45 83.33
115.38 142.86
Group 5-6 5-7 5-8 5-
9 5-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.5% acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of acetic acid (x 10-3 %) 23.81 45.45 83.33
115.38 142.86
Group 6-1 6-2 6-3 6-
4 6-5
5% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.6 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 50.29 48.00 44.00
40.62 37.7
Final concentration of acetic acid (x 10-3 %) 28.57 54.55 100.00
138.46 171.43
Group 6-6 6-7 6-8 6-
9 6-10
1% DCA-Na solution (mL) 1.00 1.00 1.00
1.00 1.00
0.6 % acetic acid (mL) 0.05 0.10 0.20
0.30 0.40
Final concentration of DCA-Na (mg/mL) 10.06 9.60 8.80
8.12 7.54
Final concentration of acetic acid (x 10-3 %) 28.57 54.55 100.00
138.46 171.43
[089] Acetic acid solutions with concentration higher than 57.14 x 10-3 %
precipitated after
added to 1% DCA-Na solution (<8.80 mg/mL); acetic acid solutions with
concentration higher
than 45.45 x 10-3 % precipitated after added to 1% DCA-Na solution (29.60
mg/mL) (FIG.
10b-f). Acetic acid solutions with concentration higher than 171.43 x 10-3 %
precipitated after
added to 5% DCA-Na solution (<37.7 mg/mL); acetic acid solutions with
concentration higher
than 100.00 x 10-3 % precipitated after added to 5% DCA-Na solution (244.00
mg/mL) (FIG.
10d-f). Mixtures of DCA-Na and acetic acid with higher concentration of acetic
acid started to
CA 03220724 2023- 11- 28
form gel around 20 minutes when placed at 25 C, while mixtures placed at 37 or
42 C formed
suspensions (or precipitations) within a short period of time (FIG. 10b-f).
Mixtures of 1%
DCA-Na and acetic acid added to 0.9% saline formed gel around 20 minutes at
acetic acid
concentration 246.15 x 10-3 % (FIG. 10b-e). Mixtures of 5% DCA-Na and acetic
acid added to
0.9% saline formed gel around 20 minutes at acetic acid concentration? 92.30 x
10-3 % (FIG.
10d-e).
10901 In Example 8, the compositions added with 0.9% saline can form gel when
the final
concentration of DCA-Na was 7.54-40.62 mg/mL, and the final concentration of
acetic acid was
46.15-142.86 x 10-3 %.
10911 The present invention demonstrated that cytolytic compound, especially
deoxycholic
acid, or its salt DCA-Na could form a slow-releasing gel, gel-forming solution
or gel-forming
suspension after mixing with amino acid (or cationic ion) at low pH or organic
acid. Additional
non-inflammatory drugs, such as lysine aspirin and dexamethasone sodium
phosphate, and local
anesthetic lidocaine could be added to the formulation of DCA-Na gel to reduce
local
inflammation. The present invention provides compositions of slow-releasing
cytolytic
compound, such as deoxycholic acid or its salt in gel or gel-forming solution
(or suspension) for
reduction of fat and with the addition of anti-inflammatory drugs and/or local
anesthetic for the
non-surgical reduction or removal of localized fat with reduced inflammation
or other adverse
effects and shorten the interval between each treatment and the whole
treatment process. The
injectable composition of the invention may optionally comprise saline, and
may be in the form
of gel during or after injection.
CA 03220724 2023- 11- 28
